Sexual attraction with pollination during feeding behaviour: implications for transitions between specialized strategies.

BACKGROUND AND AIMS: Understanding the origin of pollination by sexual deception has proven challenging, as sexually deceptive flowers are often highly modified, making it hard to resolve how any intermediate forms between sexual deception and an ancestral strategy might have functioned. Here, we report the discovery in Caladenia (Orchidaceae) of sexual attraction with pollination during feeding behaviour, which may offer important clues for understanding shifts in pollination strategy. METHODS: For Caladenia robinsonii, we observed the behaviour of its male wasp pollinator, Phymatothynnus aff. nitidus (Thynnidae), determined the site of release of the sexual attractant, and experimentally evaluated if the position of the attractant influences rates of attempted copulation and feeding behaviour. We applied GC-MS to test for surface sugar on the labellum. To establish if this pollination strategy is widespread in Caladenia, we conducted similar observations and experiments for four other Caladenia species. KEY RESULTS: In C. robinsonii, long-range sexual attraction of the pollinator is via semiochemicals emitted from the glandular sepal tips. Of the wasps landing on the flower, 57 % attempted copulation with the sepal tips, while 27 % attempted to feed from the base of the labellum, the behaviour associated with pollen transfer. A similar proportion of wasps exhibited feeding behaviour when the site of odour release was manipulated. A comparable pollination strategy occurs in another phylogenetically distinct clade of Caladenia. CONCLUSIONS: We document a previously overlooked type of sexual deception for orchids involving long-distance sexual attraction, but with pollination occurring during feeding behaviour at the labellum. We show this type of sexual deception operates in other Caladenia species and predict that it is widespread across the genus. Our findings may offer clues about how an intermediate transitional strategy from a food-rewarding or food-deceptive ancestor operated during the evolution of sexual deception.

Detecting Pathogenic Phytophthora Species Using Volatile Organic Compounds.

There are several highly damaging Phytophthora species pathogenic to forest trees, many of which have been spread beyond their native range by the international trade of live plants and infested materials. Such introductions can be reduced through the development of better tools capable of the early, rapid, and high-throughput detection of contaminated plants. This study utilized a volatilomics approach (solid-phase microextraction coupled to gas chromatography-mass spectrometry) to differentiate between several Phytophthora species in culture and discriminate between healthy and Phytophthora-inoculated European beech and pedunculate oak trees. We tentatively identified 14 compounds that could differentiate eight Phytophthora species from each other in vitro. All of the Phytophthora species examined, except Phytophthora cambivora, uniquely produced at least one compound not observed in the other species; however, most detected compounds were shared between multiple species. Phytophthora polonica had the most unique compounds and was the least similar of all the species examined. The inoculated seedlings had qualitatively different volatile profiles and could be distinguished from the healthy controls by the presence of isokaurene, anisole, and a mix of three unknown compounds. This study supports the notion that volatiles are suitable for screening plant material, detecting tree pathogens, and differentiating between healthy and diseased material.

The volatile chemistry of orchid pollination.

Covering: up to September 2022Orchids are renowned not only for their diversity of floral forms, but also for their many and often highly specialised pollination strategies. Volatile semiochemicals play a crucial role in the attraction of a wide variety of insect pollinators of orchids. The compounds produced by orchid flowers are as diverse as the pollinators they attract, and here we summarise some of the chemical diversity found across orchid taxa and pollination strategies. We focus on compounds that have been experimentally demonstrated to underpin pollinator attraction. We also highlight the structural elucidation and synthesis of a select subset of important orchid pollinator attractants, and discuss the ecological significance of the discoveries, the gaps in our current knowledge of orchid pollination chemistry, and some opportunities for future research in this field.

Drakolide Structure-activity Relationships for Sexual Attraction of Zeleboria Wasp Pollinator.

Orchids pollinated by sexual deception lure their specific male pollinators by sex pheromone mimicry. Despite the growing list of chemically diverse semiochemicals known to be involved, the chemical basis and flexibility of this extreme pollinator specificity are not fully understood. One promising but rarely applied tool is the synthesis and field testing of chemically related variants for investigating the structural specificity of the pheromone mimics. Here, we build on the discovery of the unusual semiochemical blend used by Drakaea micrantha to sexually lure its male Zeleboria thynnine wasp pollinator. This blend consists of a ß-ketolactone (drakolide) and two specific hydroxymethylpyrazines, presumably drawn from two distinct biosynthetic pathways. Here, we synthesized and tested the activity of various stereo- and structural isomers of the naturally occurring drakolide. Our study confirmed that in blends with the two pyrazines, both a mixture of stereoisomers, and the specific stereoisomer of the natural drakolide, elicit high rates of landings and attempted copulations. However, in the absence of pyrazines, both the number of responses and the level of sexual attraction were significantly reduced. When structural analogs were substituted for the natural drakolide, attractiveness and degree of sexual behaviour varied but were generally reduced. Based on our findings, and prior knowledge that related hydroxymethylpyrazines are active in other Drakaea spp., we conclude that the dual sex pheromone mimicry of D. micrantha likely evolved via initial changes in just one of the two biosynthetic pathways. Most plausibly, this involved modifications in the drakolides, with the pyrazines as a 'pre-adaption' enhancing the sexual response.

Identification of (Z)-8-Heptadecene and n-Pentadecane as Electrophysiologically Active Compounds in Ophrys insectifera and Its Argogorytes Pollinator.

Sexually deceptive orchids typically depend on specific insect species for pollination, which are lured by sex pheromone mimicry. European Ophrys orchids often exploit specific species of wasps or bees with carboxylic acid derivatives. Here, we identify the specific semiochemicals present in O. insectifera, and in females of one of its pollinator species, Argogorytes fargeii. Headspace volatile samples and solvent extracts were analysed by GC-MS and semiochemicals were structurally elucidated by microderivatisation experiments and synthesis. (Z)-8-Heptadecene and n-pentadecane were confirmed as present in both O. insectifera and A. fargeii female extracts, with both compounds being found to be electrophysiologically active to pollinators. The identified semiochemicals were compared with previously identified Ophrys pollinator attractants, such as (Z)-9 and (Z)-12-C27-C29 alkenes in O. sphegodes and (Z)-9-octadecenal, octadecanal, ethyl linoleate and ethyl oleate in O. speculum, to provide further insights into the biosynthesis of semiochemicals in this genus. We propose that all these currently identified Ophrys semiochemicals can be formed biosynthetically from the same activated carboxylic acid precursors, after a sequence of elongation and decarbonylation reactions in O. sphegodes and O. speculum, while in O. insectifera, possibly by decarbonylation without preceding elongation.

A Specific Blend of Drakolide and Hydroxymethylpyrazines: An Unusual Pollinator Sexual Attractant Used by the Endangered Orchid Drakaea micrantha.

Bioactive natural products underpin the intriguing pollination strategy used by sexually deceptive orchids. These compounds, which mimic the sex pheromones of the female insect, are emitted in particular blends to lure male insect pollinators of specific species. By combining methods from field biology, analytical chemistry, electrophysiology, crystallography, and organic synthesis, we report that an undescribed ß-hydroxylactone, in combination with two specific hydroxymethylpyrazines, act as pollinator attractants in the rare hammer orchid Drakaea micrantha. This discovery represents an unusual case of chemically unrelated compounds being used together as a sexual attractant. Furthermore, this is the first example of the identification of pollinator attractants in an endangered orchid, enabling the use of chemistry in orchid conservation. Our synthetic blend is now available to be used in pollinator surveys to locate suitable sites for plant conservation translocations.

2-(Tetrahydrofuran-2-yl)acetic Acid and Ester Derivatives as Long-Range Pollinator Attractants in the Sexually Deceptive Orchid Cryptostylis ovata.

Sexually deceptive orchids achieve pollination by luring male insects to flowers through chemical and sometimes visual mimicry of females. An extreme example of this deception occurs in Cryptostylis, one of only two genera where sexual deception is known to induce pollinator ejaculation. In the present study, bioassay-guided fractionations of Cryptostylis solvent extracts in combination with field bioassays were implemented to isolate and identify floral volatiles attractive to the pollinator Lissopimpla excelsa. ( S)-2-(Tetrahydrofuran-2-yl)acetic acid [( S)-1] and the ester derivatives methyl ( S)-2-(tetrahydrofuran-2-yl)acetate [( S)-2] and ethyl ( S)-2-(tetrahydrofuran-2-yl)acetate [( S)-3], all previously unknown semiochemicals, were confirmed to attract L. excelsa males in field bioassays. Chiral-phase GC and HPLC showed that the natural product 1 comprised a single enantiomer, its S-configuration being confirmed by synthesis of the two enantiomers from known enantiomers of tetrahydrofuran-2-carboxylic acid.

Structure-Activity Studies of Semiochemicals from the Spider Orchid Caladenia plicata for Sexual Deception.

Sexually deceptive orchids attract specific pollinators by mimicking insect sex pheromones. Normally this mimicry is very specific and identical compounds have been identified from orchids and matching females of the pollinators. In this study, we conduct a detailed structure-activity investigation on isomers of the semiochemicals involved in the sexual attraction of the male pollinator of the spider orchid Caladenia plicata. This orchid employs an unusual blend of two biosynthetically unrelated compounds, (S)-ß-citronellol and 2-hydroxy-6-methylacetophenone, to lure its Zeleboria sp. thynnine wasp pollinator. We show that the blend is barely attractive when (S)-ß-citronellol is substituted with its enantiomer, (R)-ß-citronellol. Furthermore, none of the nine-possible alternative hydroxy-methylacetophenone regioisomers of the natural semiochemical are active when substituted for the natural 2-hydroxy-6-methylacetophenone. Our results were surprising given the structural similarity between the active compound and some of the analogues tested, and results from previous studies in other sexually deceptive orchid/wasp systems where substitution with analogues was possible. Interestingly, high-level ab initio and density functional theory calculations of the hydroxy-methylacetophenones revealed that the active natural isomer, 2-hydroxy-6-methylacetophenone, has the strongest intramolecular hydrogen bond of all regioisomers, which at least in part may explain the specific activity.

The Spider Orchid Caladenia crebra Produces Sulfurous Pheromone Mimics to Attract its Male Wasp Pollinator.

One of the most intriguing natural observations is the pollination of orchids by sexual deception. Chemicals underpin this interaction between the orchid and its sexually attracted male insect pollinator, with the signaling compounds involved, called semiochemicals, predicted to mimic the chemical composition of the sex pheromone. We identified floral semiochemicals from Caladenia (spider orchids) for the first time. We further demonstrate that C. crebra attracts its single pollinator species with a unique system of (methylthio)phenols, three of which are new natural products. Furthermore, as predicted, the same compounds constitute the sex pheromone of the pollinator, the thynnine wasp Campylothynnus flavopictus, representing the first occurrence of sulfurous sex pheromones in Hymenoptera.

Parapheromones for Thynnine Wasps.

Sexually deceptive orchids produce floral volatiles that attract male insect pollinators. This interaction between flower and pollinator normally is highly specific. In the few cases where the chemical composition of the volatiles is known, the compounds have been found to be identical to those that comprise the sex pheromone of the female wasp. In this study, we investigated whether there is potential for flexibility in the molecular structure of the chemical cues used to mediate these specific interactions. Specifically, we asked whether strong sexual attraction can be maintained with structural modifications of sex pheromone components. Our study focused on the orchid, Drakaea glyptodon, which is pollinated by males of the thynnine wasp, Zaspilothynnus trilobatus. Three alkylpyrazines and a unique hydroxymethylpyrazine are components of the female produced sex pheromone of Z. trilobatus, and also the semiochemicals produced by the orchid that lures the males as pollinators. A blend of 2-butyl-3,5-dimethylpyrazine and 2-hydroxymethyl-3,5-diethyl-6-methylpyrazine (3:1) is as attractive as the full blend of four compounds. Therefore, in this study we substituted 2-hydroxymethyl-3,5-diethyl-6-methylpyrazine with one of five structurally related parapheromones in a blend with 2-butyl-3,5-dimethylpyrazine. All blends tested stimulated approaches by male wasps, with some also eliciting landing and attempted copulation. High-level calculations (G4(MP2)) showed the energy differences between the structural isomers were small, although the degree of sexual attraction varied, indicating the importance of structural factors for activity. One of the parapheromones, 2-hydroxymethyl-3,5-dimethyl-6-ethylpyrazine, elicited similar proportions of approaches, landings, and attempted copulations as the sex pheromone at the ratio and dose tested. The findings suggest that there is potential for chemical flexibility in the evolution of sexual deception.

Discovery of pyrazines as pollinator sex pheromones and orchid semiochemicals: implications for the evolution of sexual deception.

Sexually deceptive orchids employ floral volatiles to sexually lure their specific pollinators. How and why this pollination system has evolved independently on multiple continents remains unknown, although preadaptation is considered to have been important. Understanding the chemistry of sexual deception is a crucial first step towards solving this mystery. The combination of gas chromatography-electroantennographic detection (GC-EAD), GC-MS, synthesis and field bioassays allowed us to identify the volatiles involved in the interaction between the orchid Drakaea glyptodon and its sexually attracted male thynnine wasp pollinator, Zaspilothynnus trilobatus. Three alkylpyrazines and one novel hydroxymethyl pyrazine were identified as the sex pheromone of Z. trilobatus and are also used by D. glyptodon for pollinator attraction. Given that our findings revealed a new chemical system for plants, we surveyed widely across representative orchid taxa for the presence of these compounds. With one exception, our chemical survey failed to detect pyrazines in related genera. Collectively, no evidence for preadaptation was found. The chemistry of sexual deception is more diverse than previously known. Our results suggest that evolutionary novelty may have played a key role in the evolution of sexual deception and highlight the value of investigating unusual pollination systems for advancing our understanding of the role of chemistry in evolution.

Practical preparation of unsaturated very-long-chain fatty acids (VLCFAs) and very-long-chain alkene pollinator attractants.

To prepare very-long-chain fatty acids and alkenes (VLCFAs and VLC alkenes) that are known pollinator attractants for sexually deceptive orchids, and biosynthetic precursors thereof, we applied a methodology allowing us to prepare monounsaturated VLCFAs with chain lengths up to 28 carbons and VLC alkenes up to 31 carbons. We implemented a coupling reaction between commercially available terminal alkynes and bromoalkanoic acids to prepare VLCFAs, allowing the products to be formed in two steps. For VLC alkenes, with many alkyltriphenylphosphonium bromides commercially available, we applied a Wittig reaction approach to prepare (Z)-configured monoenes in a single step. Using practical methods not requiring special reagents or equipment, we obtained 11 VLCFAs in > 90% isomeric purity, and 17 VLC alkenes in > 97% isomeric purity. Such general and accessible synthetic methods are essential for chemical ecology and biochemistry research to aid researchers in unambiguously identifying isolated semiochemicals and their precursors.

Odour-mediated oviposition site selection in Aedes aegypti depends on aquatic stage and density.

BACKGROUND: Olfaction plays an important role in the selection and assessment of oviposition sites by mosquitoes. Volatile organic compounds (VOCs) associated with potential breeding sites affect the behaviour of gravid mosquitoes, with VOCs from aquatic stages of conspecific mosquitoes influencing and regulating oviposition. The purpose of this study was to conduct a systematic analysis of the behavioural response of gravid Aedes aegypti to conspecific aquatic stage-conditioned water, to identify the associated bioactive VOCs and to determine how blends of these VOCs regulate oviposition site selection and stimulate egg-laying. METHODS: Using a multi-choice olfactory oviposition assay, controlling for other sensory modalities, the responses of individual females to water conditioned with different densities of conspecific aquatic stages were assessed. The conditioned water samples from the most preferred density of each aquatic stage were subsequently compared to each other using the same oviposition assay and analysed using an analysis of variance (ANOVA) followed by a Tukey post-hoc test. Using combined gas chromatography and electroantennographic detection or mass spectrometry, bioactive VOCs from the preferred density of each aquatic stage were identified. Synthetic blends were prepared based on the identified ratios of bioactive VOCs in the aquatic stages, and then tested to determine the oviposition choice of Ae. aegypti in a dose-dependent manner, against a solvent control, using a dual-choice assay. This dataset was analysed using nominal logistic regression followed by an odds ratio comparison. RESULTS: Gravid Ae. aegypti responded stage- and density-dependently to water conditioned with eggs, second- and fourth-instar larvae, and pupal exuviae, but not to water conditioned with pupae alone. Multi-choice assays demonstrated that gravid mosquitoes preferred to oviposit in water conditioned with fourth-instar larvae, over the other aquatic stage-conditioned water. Gravid Ae. aegypti were attracted, and generally stimulated, to oviposit in a dose-dependent manner to the individual identified synthetic odour blends for the different aquatic stages. CONCLUSIONS: Intraspecific VOCs regulate oviposition site selection in Ae. aegypti in a stage- and density-dependent manner. We discuss the need for further studies to evaluate the identified synthetic blends to modulate the odour-mediated oviposition of Ae. aegypti under field conditions.

Determining the parent and associated fragment formulae in mass spectrometry via the parent subformula graph.

BACKGROUND: Identifying the molecular formula and fragmentation reactions of an unknown compound from its mass spectrum is crucial in areas such as natural product chemistry and metabolomics. We propose a method for identifying the correct candidate formula of an unidentified natural product from its mass spectrum. The method involves scoring the plausibility of parent candidate formulae based on a parent subformula graph (PSG), and two possible metrics relating to the number of edges in the PSG. This method is applicable to both electron-impact mass spectrometry (EI-MS) and tandem mass spectrometry (MS/MS) data. Additionally, this work introduces the two-dimensional fragmentation plot (2DFP) for visualizing PSGs. RESULTS: Our results suggest that incorporating information regarding the edges of the PSG results in enhanced performance in correctly identifying parent formulae, in comparison to the more well-accepted "MS/MS score", on the 2016 Computational Assessment of Small Molecule Identification (CASMI 2016) data set (76.3 vs 58.9% correct formula identification) and the Research Centre for Toxic Compounds in the Environment (RECETOX) data set (66.2% vs 59.4% correct formula identification). In the extension of our method to identify the correct candidate formula from complex EI-MS data of semiochemicals, our method again performed better (correct formula appearing in the top 4 candidates in 20/23 vs 7/23 cases) than the MS/MS score, and enables the rapid identification of both the correct parent ion mass and the correct parent formula with minimal expert intervention. CONCLUSION: Our method reliably identifies the correct parent formula even when the mass information is ambiguous. Furthermore, should parent formula identification be successful, the majority of associated fragment formulae can also be correctly identified. Our method can also identify the parent ion and its associated fragments in EI-MS spectra where the identity of the parent ion is unclear due to low quantities and overlapping compounds. Finally, our method does not inherently require empirical fitting of parameters or statistical learning, meaning it is easy to implement and extend upon. SCIENTIFIC CONTRIBUTION: Developed, implemented and tested new metrics for assessing plausibility of candidate molecular formulae obtained from HR-MS data.

Critical Pollination Chemistry: Specific Sesquiterpene Floral Volatiles in Carrot Inhibit Honey Bee Feeding.

Many plants rely on insect pollination, yet numerous agricultural plant-breeding programs focus on traits that appeal to growers and consumers instead of pollinators, leading to declining pollinator attraction and crop yields. Using hybrid carrot seed production as a model, we investigated low-yielding carrot varieties by analyzing sugars and minerals in nectar and floral volatile composition. While the analysis of nectar sugars and minerals did not reveal any key differences between the carrot varieties, differences between the 112 detected volatiles in 23 samples were observed. Numerous differentiating sesquiterpenes were identified in floral solvent extracts, and subsequent behavioral assays showed that ß-ocimene from higher-yielding carrot varieties stimulated nectar feeding (attractant), while α- and ß-selinene from lower-yielding lines decreased feeding (deterrents). Sesquiterpenes have previously been implicated in plant defense, suggesting a trade-off between pollination and protection. Our results highlight the importance of volatiles as regulators of pollinator attraction in agricultural settings.

Discovery of tetrasubstituted pyrazines as semiochemicals in a sexually deceptive orchid.

Sexually deceptive orchids employ mimicry of insect sex pheromones to exploit a diverse group of pollinators. The chemical structures of five semiochemicals (1-3, 7, 8) produced by populations of the warty hammer orchid, Drakaea livida, pollinated by a thynnine wasp in the genus Catocheilus were elucidated. With the exception of (2,5-dimethylpyrazin-3-yl)methyl 3-methylbutanoate (7), all active compounds were tetrasubstituted pyrazines, including hydroxymethyl (1) and ester (2 and 3) trimethylpyrazine derivatives. Male Catocheilus wasps were responsive to all of these compounds in GC-EAD experiments.

Seed dispersal: Hungry hornets are unexpected and effective vectors.

A new study finds that, in the forests of tropical China, hungry hornets are lured to the fruits of Aquilaria sinensis by highly volatile compounds structurally similar to volatiles from herbivore-damaged leaves. The hornets disperse the short-lived seeds rapidly to optimal new habitats.

Three Chemically Distinct Floral Ecotypes in Drakaea livida, an Orchid Pollinated by Sexual Deception of Thynnine Wasps.

Sexually deceptive orchids are unusual among plants in that closely related species typically attract different pollinator species using contrasting blends of floral volatiles. Therefore, intraspecific variation in pollinator attraction may also be underpinned by differences in floral volatiles. Here, we tested for the presence of floral ecotypes in the sexually deceptive orchid Drakaea livida and investigated if the geographic range of floral ecotypes corresponded to variation in pollinator availability. Pollinator choice trials revealed the presence of three floral ecotypes within D. livida that each attracts a different species of thynnine wasp as a pollinator. Surveys of pollinator distribution revealed that the distribution of one of the ecotypes was strongly correlated with that of its pollinator, while another pollinator species was present throughout the range of all three ecotypes, demonstrating that pollinator availability does not always correlate with ecotype distribution. Floral ecotypes differed in chemical volatile composition, with a high degree of separation evident in principal coordinate analysis. Some compounds that differed between ecotypes, including pyrazines and (methylthio)phenols, are known to be electrophysiologically active in thynnine wasp antennae. Based on differences in pollinator response and floral volatile profile, the ecotypes represent distinct entities and should be treated as such in conservation management.

Utilizing volatile organic compounds for early detection of Fusarium circinatum.

Fusarium circinatum, a fungal pathogen deadly to many Pinus species, can cause significant economic and ecological losses, especially if it were to become more widely established in Europe. Early detection tools with high-throughput capacity can increase our readiness to implement mitigation actions against new incursions. This study sought to develop a disease detection method based on volatile organic compound (VOC) emissions to detect F. circinatum on different Pinus species. The complete pipeline applied here, entailing gas chromatography-mass spectrometry of VOCs, automated data analysis and machine learning, distinguished diseased from healthy seedlings of Pinus sylvestris and Pinus radiata. In P. radiata, this distinction was possible even before the seedlings became visibly symptomatic, suggesting the possibility for this method to identify latently infected, yet healthy looking plants. Pinus pinea, which is known to be relatively resistant to F. circinatum, remained asymptomatic and showed no changes in VOCs over 28 days. In a separate analysis of in vitro VOCs collected from different species of Fusarium, we showed that even closely related Fusarium spp. can be readily distinguished based on their VOC profiles. The results further substantiate the potential for volatilomics to be used for early disease detection and diagnostic recognition.

Pathogenic fungus uses volatiles to entice male flies into fatal matings with infected female cadavers.

To ensure dispersal, many parasites and pathogens behaviourally manipulate infected hosts. Other pathogens and certain insect-pollinated flowers use sexual mimicry and release deceptive mating signals. However, it is unusual for pathogens to rely on both behavioural host manipulation and sexual mimicry. Here, we show that the host-specific and behaviourally manipulating pathogenic fungus, Entomophthora muscae, generates a chemical blend of volatile sesquiterpenes and alters the profile of natural host cuticular hydrocarbons in infected female housefly (Musca domestica) cadavers. Healthy male houseflies respond to the fungal compounds and are enticed into mating with female cadavers. This is advantageous for the fungus as close proximity between host individuals leads to an increased probability of infection. The fungus exploits the willingness of male flies to mate and benefits from altering the behaviour of uninfected male host flies. The altered cuticular hydrocarbons and emitted volatiles thus underlie the evolution of an extended phenotypic trait.